The human peptide hormone insulin controls blood glucose levels during feeding and fasting and acts via cell surface receptors of liver and adipose tissue cells. Besides controlling uptake, storage and production of glucose, insulin is also i.a. involved in the control of production and breakdown of lipids.
Deficiencies in the supply of insulin result in elevated blood glucose concentrations (hyperglycaemia) and, in chronic form, are revealed by the classic symptoms of diabetes mellitus (DM). Insulin is administered daily to patients suffering from DM.
Mature human insulin is a peptide that is comprised of an A (alpha) and a B (beta) chain, linked by 2 inter-chain disulfide bridges. A third disulfide bridge connects two residues of the A chain. In proinsulin, the biosynthetic precursor, the A and B chains, are connected to each other by the C peptide, which role is to aid in appropriate disulfide bridge formation between the A and B segments and to allow proper folding of the proinsulin molecule. In the last stage of maturation, proteolytic enzymes cleave at specific amino acid residues to release the C peptide thus forming the mature insulin.
Biosynthetic recombinant human insulin is presently i.a. manufactured as proinsulin dike polypeptides expressed in e.g. E. coli or yeast (see e.g. U.S. Pat. No. 5,593,860). In most cases, proinsulin is produced as a fusion protein or recombinant hybrid, wherein the proinsulin is cross-linked via methionine residues to a heteroprotein, such as for instance human copper/zinc superoxide dismutase (hSOD). Normally, these hybrids accumulate in the recombinant cells as intracellular precipitated protein or inclusion bodies.
During manufacturing of recombinant proinsulin, the inclusion bodies, obtained by centrifugation after lysis of the cells, are washed with a detergent or a denaturant at a low concentration. Such treatment is repeated to increase the purity of the desired protein. In order to minimize intermolecular hydrophobic interaction, and formation of incorrect disulfide bonds, the washed inclusion bodies are dissolved in a denaturant, such as a urea or guanidine-HCl solution containing a reducing agent such as dithiothreitol (DTT) or 2-mercaptoethanol, and recovered by precipitation.
The hybrid is normally isolated and cleaved by cyanogen bromide (CNBr) in order to release the proinsulin polypeptide from the heteroprotein. The proinsulin is further modified by oxidative sulfitolysis to proinsulin S-sulfonate (See e.g. EP 0 055 945 and EP 0 196 056). The proinsulin S-sulfonate is then further purified and refolded to a native conformation under reducing conditions by using reducing agents such as dithiothreitol (DTT), 2-mercaptoethanol, etc. or a redox system such as glutathione. Conversion of the proinsulin to insulin, i.e. removal of the C peptide, is achieved by the combined action of trypsin and carboxypeptidase B. Finally insulin is purified through e.g. reverse-phase high performance liquid chromatography (RP-HPLC) and optionally crystallized.
During the complete isolation procedure, from lysis of the recombinant cells through to proper folding of the proinsulin, free thiol groups of the cysteine residues comprised in the (fusion) protein, may form incorrect or aspecific intra- or intermolecular disulfide bridges. This results in scrambled peptides and inactive hormones or in the formation of ‘aggregates’ of desired proteins and contaminating proteins (U.S. Pat. No. 6,150,134). Therefore, free thiol groups should either be blocked in order to prevent the formation of incorrect disulfide bridges or procedures should involve selectively cleaving of incorrect disulfide bonds.
Disulfide bond cleaving may i.a. be achieved by: a) modifying the cysteine residues into cysteic acid by cysteic acid oxidation or performic acid treatment; b) modifying the cysteine into S-sulfo-cysteine by sulfitolysis (R—S—S—R→2R—SO3); c) reduction by means of certain reducing agents, such as phosphines or mercaptans (see e.g. EP 0 379 162).
However, preventing the incorrect disulfide bonds to form is preferred over the use of “oxido-shuffling” agents following protein isolation, and to achieve this, again several reducing agents, such as dithiotreitol (DTT), β-mercaptoethanol, cysteine, glutathione, E-mercaptoethylamine or thioglycollic acid, are most commonly used.
The use of all above-mentioned reducing agents, however, poses problems in that they pose a toxic risk, in that they are costly, and in that their contamination of the product requires additional purification.
Therefore, the conventional process for preparing recombinant proinsulin is proven to be less satisfactory since it involves complicated steps of dissolution and sulfonation, purification, concentration, wherein the refolding of the proinsulin progresses inefficiently resulting in reduced yields of the desired protein.
Accordingly, there is a need for an improved process for the isolation of proteins which comprise disulfide-bonds in their native conformation in an efficient and less contaminating manner